Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.)

GENE-39869; No. of pages: 9; 4C: Gene xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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Neelofar Mirza, Gohar Taj, Sandeep Arora, Anil Kumar ⁎

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Department of Molecular Biology & Genetic Engineering, College of Basic Sciences & Humanities, G B Pant University of Agriculture & Technology, Pantnagar 263145, Uttarakhand, India

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a r t i c l e

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Article history: Received 27 May 2014 Received in revised form 31 July 2014 Accepted 2 August 2014 Available online xxxx

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Keywords: Eleusine coracana Transcript abundance Differential expression CAX1 TPC1 ATPase Calmodulin CaM kinase Source to sink

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Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca2+/H+ antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca2+ ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca2+ ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet. © 2014 Published by Elsevier B.V.

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Most of the mineral requirement in humans can be met by plant products. In turn plants must acquire all mineral elements from the rhizosphere, through their roots and deliver them to the shoot since no gene product exists which allows plants to synthesize minerals. Thus, a complex, integrated system of tissues and membrane processes is required to move minerals from the root–soil interface to each cell throughout the plant (Grusak, 2002). Calcium like most other elements is absorbed in free ionic form by the root cells and mobilized from the roots to various vegetative and reproductive tissues. Plants rarely lack a calcium supply from the soil solution, the variation in calcium content is therefore mostly genetic (Panwar et al., 2010; White and Broadley, 2003, 2005b). The commelinoid monocots such as cereals have been reported to have lower shoot and grain calcium concentrations than eudicots (Broadley et al., 2003, 2004; Pfeiffer and McClafferty, 2007a,

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1. Introduction

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Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.)

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Abbreviations: CaMK, calmodulin dependent kinase; SAM, shoot apical meristem; qPCR, quantitative PCR; CRD, completely randomized design; CAX1, Ca2 +/H+ antiporter or exchanger; TPC1, two-pore channel; CDPK, Ca2 +-dependent protein kinase. ⁎ Corresponding author. E-mail address: [email protected] (A. Kumar).

b; Thompson et al., 1997; White and Broadley, 2005a). Distinctive patterns of mineral accumulation in plant tissues, cell types and subcellular compartments are proposed to be the product of selective transport processes catalyzing their short distance as well as long distance movement (Karley and White, 2009). The short-distance ion movement depends upon the membrane transporters while the long-distance mineral movement utilizes the xylem and phloem pathways. An appropriate transport protein must be present to facilitate movement of calcium into the root symplasm. Once it enters this compartment, calcium can either provide for the local nutritional needs of the root cells, or it can be moved to the xylem pathway (Grusak, 2002). The xylem transport system is believed to provide an open route for the bulk flow of water and mineral ions (Kramer and Boyer, 1995). Thus, calcium within the xylem sap is proposed to be delivered nonselectively as columns of water are pulled up through the plant primarily by the transpirational pull and are accumulated preferentially in those tissues with high rates of water loss viz. most leaves and certain reproductive structures, such as the pod walls (Grusak and Pomper, 1999). The non-exposed structures e.g. developing seeds can exhibit very low to negligible rate of transpiration and consequently, mineral delivery via the xylem pathway to these types of organs will be low (Ho and White, 2005; Welch, 1999; White, 2005; White and Broadley,

http://dx.doi.org/10.1016/j.gene.2014.08.005 0378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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2.1. Plant material

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Two finger millet genotypes viz. GPHCPB-1 (GP-1) and GPHCPB-45 (GP-45) differing in their grain calcium content by about 100 mg/100 g of seeds were selected. The plants were grown in 10 kg earthen pots (2:1:1; soil:sand:vermi-compost) in polyhouse (relative humidity 40%; temperature 37–40 °C) under identical conditions. Geographically, the site lies at 29°N latitude, 79.3°E longitude and an altitude of 243.8 m above the mean sea level in Tarai plains ~30 km southward to foothills of Shivalik ranges, Himalayas.

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2. Materials and methods

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2.2. Selection of various developmental stages and sample collection

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The abundance of specific mRNA species of these genes was investigated in root, stem, leaf, flag leaf and developing spike at different vegetative and reproductive stages in two finger millet genotypes, GP45 accumulating more calcium in the grain than GP1, for identification of the key transporters leading to differential accumulation. The expression of calmodulin, calmodulin dependent kinase (CaMK) genes and 14-3-3 gene was also studied in the aforesaid tissues to identify the players involved in regulation of calcium transport. The study presents a tissue wide comparison of genes involved in calcium translocation, accumulation and their possible regulation using two different genotypes.

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For qPCR analysis, root, stem and leaf tissues were collected at three different vegetative stages of 30, 60 and 90 DAS (days after sowing). Four different developmental stages of the spike, booting or inflorescence immergence, anthesis, grain filling and grain ripening or maturation were identified on the basis of morphology and development stage of ovary and anthers and were designated as S1, S2, S3 and S4 respectively (Fig. 1). Florets were removed from the spikelets and were documented using a stereo-zoom microscope (SZ-PT Olympus, Japan) after removing the lemma and palea. For Ca2+ estimation and RNA isolation, root tips (1–1.5 cm) and 1/3rd portion from the tip of the 3rd leaf were collected. For stem, samples were collected from the top (1–1.5 cm) including the shoot apical meristem (SAM) of the main tiller or culm. Spike and corresponding flag leaf samples were collected in a similar manner, collecting the 1/3rd portion from the tip. All the samples were collected in the forenoon, between 8.00 and 9.00 AM.

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2.3. Calcium content estimation in various tissues

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Atomic absorption spectrophotometer was used to estimate calcium in different tissues and mature seeds of the two genotypes. The samples were wet ashed using di-acid mixture of 3 parts concentrated (16 N) nitric acid and one part (70% w/v) perchloric acid according to Barbeau and Hilu (1993). The digested samples were dissolved in 2% HNO3. Each sample was prepared in triplicate.

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2.4. Isolation of genes involved in Ca2+ transport and regulation

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2.4.1. Primer designing The MPSS and Affymetrix gene expression data of rice were used to identify the genes of Ca2+ transporters that predominantly express during the different developmental stages of the rice plant (Goel et al., 2012). The gene sequences were retrieved from TIGR database and aligned using MegAlign module of DNASTAR. Based on consensus sequences different sets of gene specific primers were designed manually from the least conserved region to isolate the specific isoforms (Table 1). The primers were so designed to be used for quantitative PCR (qPCR) also.

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2003). These organs must acquire minerals and photosynthates via phloem and consequently are generally low in calcium (Grusak, 2002; Grusak and Pomper, 1999; Karley and White, 2009; Kramer and Boyer, 1995; Tibbitts and Palzkill, 1979). However, the presence of specific transporter proteins, provides certain direction and selectivity to calcium transport and accumulation. Calcium can enter root cells through a variety of calcium-permeable cation channels including the de-polarization activated Ca2+ channels, which may be encoded by homologs of the AtTPC1 gene (Medvedev, 2005; Peiter et al., 2005). Once in the cytoplasm, Ca2+ is maintained at submicromolar concentrations by Ca2+-ATPases, encoded by members of the type IIA-ATPase (ECA) or calmodulin (CaM)-regulated type IIB-ATPase (ACA) gene families, and Ca2 +/H+ antiporters, such as those encoded by the CAX genes (McAinsh and Pittman, 2009; Medvedev, 2005). These transporters export Ca2+ to the apoplast and into the cellular organelles i.e. endoplasmic reticulum, golgi/endosome/pre-vacuolar compartments, plastids or vacuoles (Bickerton and Pittman, 2012; Karley and White, 2009). These transporters must be tightly regulated (via CaM and/or various Ca2 +/CaM dependent protein kinases etc.), since explicit perturbations in cytosolic Ca2 + concentrations co-ordinate specific responses to many developmental and environmental stimuli (Galon et al., 2010; Medvedev, 2005; Tuteja and Mahajan, 2007; Peiter, 2005). Increased expression of ACA family type IIB Ca2+ ATPases, CAX family members, calmodulins and Ca2 + dependent/CaM interacting protein kinases has been reported in the calcium accumulating barley leaf epidermis against the remaining leaf tissue (Richardson et al., 2007). In addition, 14-3-3 proteins are a group of highly conserved proteins interacting with several target proteins including protein kinases (Camoni et al., 1998) and ion transporters (de Boer, 2002). Ion channels (TPK1 and TPC1) in the tonoplast are also subject to regulation by 14-33 proteins (Latz et al., 2007). Therefore, in the present study we have tried to explore the tissue specific function of targeted calcium transport, channel protein etc. by analyzing their expression in a spatiotemporal manner. Finger millet or Eleusine coracana belongs to Chloridoideae subfamily grown as a cereal crop in the semi-arid tropics and subtropics of the world (Fakrudin et al., 2004). The crop is highly nutritious with some millet lines having as much as 14.2% protein and particularly enrich in the essential amino acids tryptophan, cysteine and methionine (Iyengar et al., 1945–1946). The micronutrient density of finger millet is higher than rice or wheat (Rao and Deosthale, 1983), being rich in minerals such as manganese, copper, magnesium, selenium, molybdenum, phosphorus and particularly calcium (Antony and Chandra, 1998; Vadivoo et al., 1998). Seeds, tubers and fruits are generally low in calcium content (Karley and White, 2009) however, finger millet seeds contain calcium as high as 450 mg/100 g of seeds (Panwar et al., 2010) which is more than ten folds higher as compared to those of other cereal crops. Several genotypes of finger millet vary considerably in their calcium content (Barbeau and Hilu, 1993; Kumar et al., 2011; Panwar et al., 2010; Vadivoo et al., 1998) that signify the existing genetic variation which can be utilized for development of improved plant varieties. Major differences were found among genotypes with regard to calcium distribution in seed components, with highest accumulation being in the aleurone layer of seeds (Nath et al., 2013). Therefore, it becomes interesting to investigate these finger millet genotypes with differential grain calcium content for understanding the underlying molecular mechanism and to answer intriguing questions regarding the molecular mechanism(s) associated with translocation of calcium and its accumulation in grains. Do seed specific calmodulin transducers differentially regulate the grain/seed specific calcium transporters? What is the role of protein kinases in the uptake and transport of nutrients? Do genotypic variations of calcium contents are having any genetic basis related to the translocation and accumulation of calcium from source to sink in plants? In the present investigation, attempts were made to isolate various genes involved in calcium transport and regulation from finger millet in order to develop some molecular insight about these questions.

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Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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Fig. 1. Developmental stages of finger millet spike. Four different developmental stages of the spike, (a) S1 stage i.e. booting or inflorescence immergence when florets are compact, androecium and gynoecium are very small, closely arranged and pale in color, (b) S2 stage i.e. anthesis, when flowering is half way, florets are clear; anthers and feathery stigma are visible at the tip of the florets; anthers, filament, stigma and style are clear and increase in size; anthers appear yellowish due to pollen grains and ovary is swollen, (c) S3 stage i.e. grain filling when increase in solids in the liquid endosperm is notable while crushing the caryopsis between fingers and (d) S4 stage i.e. grain ripening or maturation when 50% of spikelets are ripened and the caryopsis is hard enough so that it is difficult to divide by thumb-nail, were selected for studying the expression of the potential transporters and their regulatory genes.

Table 1 List of primer sequences for the target genes.

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CAX1

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TPC1

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CaMK1

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CaMK2

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Tubulin

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14-3-3

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Type IIB ATPase

Primer sequences (5′ to 3′)

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F-TAGAGGGTGCTCCTTCTTGC R-TCGTGTTGCACTCTTCGTTC F-TCAAGACGCACCGCCAGCTGTT R-GTGCCGACGACGTACTCGGAGA F-TCAGGGAACTGCGAATGTGTGCT R-GTTACATAAGCTAGCCAGCTTGC F-TGGCTCCTGAAGTGCTGAAGAGA R-GGGTCAGGCTGAAGCATCTGACG F-GCTTCAAAGGCTTGCATGGAACG R-TACTTGGGTGCCTCCCTCAG F-ATGATCAATGAGGTTGATGCTG R-TCCTCATCGGTTAGCTTCTCTC F-CTCCAAGCTTTCTCCCTCCT R-GCATCATCACCTCCTCCAAT F-TACGAGGAGATGGTCGAGTTC R-GAGCTCGGTCTCGATCTTG

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AB922824

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AB922825

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AB894550

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AB922826

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AB859070

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CX265249

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AB645769

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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2.4.3. Cloning, sequencing and in silico analysis The amplicons obtained by PCR were eluted from gel, cloned in 216 pGEM-T Easy cloning vector (Promega) and confirmed clones were se217 quenced using Applied Biosystems 370 at South Campus, University of 218 Delhi. The partial sequences obtained were annotated using BLASTn 219 and BLASTp (Altschul et al., 1990). The putative CDS were translated 220 to protein sequence using the ExPASyTranslate tool (http://ca.expasy. 221 org/tools/dna.html). ATPase, CAX1, TPC1, and CaM dependent kinase 222 protein sequences from different crop species, such as wheat, rice, bar223 ley, maize, and sorghum were retrieved from NCBI and aligned with fin224 ger millet isolated sequences using ClustalW (Thompson et al., 1997) 225 and phylogenetic tree was inferred using the Neighbor-Joining (NJ) 226 Q10 method of MEGA version 4.1 (Tamura et al., 2011).

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Real time qPCR was carried out using the Maxima™ SYBR Green/ ROX qPCR Master mix (2×; Fermentas) according to the manufacturer's instructions in Eppendorf Thermocycler ep Realplex 4. Two-step realtime PCR was carried out with cDNA prepared as mentioned earlier from different developmental stages of finger millet using gene specific primers given in Table 1. The reverse transcription efficiencies of selected genes and tubulin gene were almost equal as analyzed by comparing the CT values at different dilutions of cDNA (Livak and Schmitten, 2001). The following amplification program was used: 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s; 95 °C for 15 s; 60 °C for 15 s and 95 °C for 15 s. All samples were amplified in triplicate, and the mean value was considered. The relative value obtained for quantitation was expressed as 2−ΔΔCT (Livak and Schmitten, 2001).

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The samples were amplified in triplicate and mean and standard error values were calculated to plot heat map depicting the relative expression using the software ‘R’ version 2.15.2. Completely randomized design (CRD) was used for analyzing the real time data respectively. The real-time quantitative PCR data was analyzed as a four factorial analysis of variance (ANOVA) with genotype (GP-1 & GP-45), developmental stages (30 DAS, 60 DAS, 90 DAS, S1, S2, S3 & S4), tissue (root, stem, leaf, flag leaf and developing spike) and gene as factors. Correlation coefficients were also measured for various real time and calcium content data.

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3.1. Molecular cloning and annotation of genes from finger millet involved in calcium transport and accumulation

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Calcium is accumulated by plants variably among species, genotypes and even different tissues. Differential expression and regulation of various calcium transporters could possibly cause this variation in calcium accumulation (Richardson et al., 2007). The present investigation helps us to understand the molecular mechanism underlying the differential uptake, transport and accumulation of calcium and its regulation leading to elevated calcium in crop plants. Such a tissue wide gene expression study is significant in designing a rational approach to spatiotemporally control the transgene and enrich for target elements as previous studies that have constitutively over expressed genes have led to agronomic penalties (Park et al., 2005). Expression pattern of individual genes in various tissues of finger millet genotypes is shown in the heat map (Fig. 2b, d). The four factorial statistical analysis of real time data reveals that the changes in transcript level are highly significant (p b 0.01) for all the genes in each tissue. Genotype, tissue and stage, all affected the expression patterns significantly. The expression patterns of transporter genes correlated with the calcium content in various tissues. Wide spatial variation in calcium content was observed in all the tissues at vegetative and reproductive stages. However, the variation was greater in the vegetative tissues. The root and stem tissues accumulated less calcium than the leaf tissue. In reproductive stages, a continuous increase in calcium content was observed in the developing spikes in both the genotypes however, calcium was higher in GP-45 at all stages. The flag leaf was observed to accumulate more calcium than the developing spikes. The grain calcium content of the two genotypes has been determined experimentally and was found to differ by ~100 mg. GP-1 is a low calcium accumulating genotype (126.1 mg/100 g of seeds) whereas GP45 is a high calcium genotype (250.7 mg/100 g of seeds). This difference in grain calcium content among the two genotypes provides excellent plant material to study the differential accumulation of calcium.

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3.2.1. In root tissue The increased expression of TPC1, ATPase and CAX1 from 30 DAS to 90 DAS in both the genotypes is suggestive of their respective role in uptake, translocation and accumulation of calcium. The expression of these

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3.2. Differential spatial accumulation of calcium and transcript profiling of 285 isolated genes in two genotypes of finger millet 286

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preferential accumulation of the minerals in some tissues. The role and mechanism, for preferential accumulation of elements are currently unknown but may be, in part, linked to maximizing elemental availability to the plant (Conn and Gilliham, 2010; Grusak, 2002; Karley and White, 2009; White, 2001; White and Broadley, 2003). The genetic variability in the distribution of minerals in different plants and within the edible tissues has long been thought to be utilized in biofortification strategies (Conn et al., 2012; White and Broadley, 2009). In the present investigation, finger millet, accumulating high calcium in seeds, has been taken as a model system to investigate the molecular basis of elevated calcium in the cereal crop. Hence, attempts were made to identify and clone various Ca transporter genes involved in the uptake, transport and accumulation of Ca and genes involved in the regulation of these Ca transporters. Their transcript profiles were studied through real time PCR, in different tissues at various developmental stages of two finger millet genotypes differing in Ca content. A CaM stimulated type IIB Ca2 + pump (ATPase), the co-transporter Ca2 +/H+ antiporter or exchanger (CAX1), two-pore channel (TPC1) and two calmodulin dependent protein kinase (CaMK) designated as EcCaMK1 and EcCaMK2 were isolated from finger millet using gene specific primers. The partial sequences were BLAST searched to locate homologous sequences in the TIGR database. The homology was confirmed through multiple sequence alignment using ClustalW. The transporter sequences were submitted to DDBJ (Table 1). The sequences were translated using ExPASy translate tool and phylogenetic trees were inferred. The phylogenetic analysis grouped all the genes isolated from finger millet with Poaceae family.

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2.4.2. Total RNA isolation and cDNA synthesis and PCR amplification Total RNA was isolated from the various tissues of the two genotypes using the iRIS total RNA isolation kit (developed by Institute of Himalayan Bioresource Technology, Palampur, India) according to the manufacturer's instructions. DNase (Fermentas) treated total RNA (2 μg) was used to synthesize first strand cDNA by using oligo(dT) 18 primer with RevertAid H Minus M-MuLV RT (Fermentas). cDNA was used to amplify the genes using gene specific primers (Table 1). For expression analysis, minimum of three plant replicates were selected and the RNA was pooled. The finger millet tubulin gene specific primers were used with total cellular cDNA as templates in a separate tube as a positive control. The negative control in the PCR amplification included everything that was present in the tubulin gene amplification except cDNA templates.

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Research has been going on for decades now to elucidate the transport and accumulation of various minerals by plants, but still our knowledge base is lacking in the area of mineral loading and unloading and

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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Fig. 2. Expression analysis of CAX1, TPC1, ATPase, CaM, CaMK1, CaMK2 and 14-3-3 genes and calcium content in root, stem and leaf at three different vegetative stages and spike and flag leaf at four different reproductive stages viz. S1, S2, S3 and S4 of two finger millet genotypes viz. GPHCPB 1 and GPHCPB 45 differing in Ca content. b & d: heat map showing the expression levels of genes in two finger millet genotypes. The heat map was generated using the software “R” version 2.15.2. a & c: calcium content in the respective tissues of vegetative and reproductive stages.

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genes was higher in GP-45 roots corresponding to its higher calcium content (Fig. 2c) as compared to those in GP-1. The expression of all three genes in GP-1 was observed to be highest at 60 DAS (Fig. 2d). This might be due to the sudden increase in height in GP-1 genotype at 60 DAS and increased demand of calcium for the growth and differentiation of cells. In GP-45, the highest expression for these genes was observed at 90 DAS. This might correspond to the increased demand of calcium by the developing reproductive tissues in the early flowering (90 DAS) genotype GP-45. CAX1 actively pumps calcium from the cytoplasm into the vacuole. An increasing expression of ATPase and CAX1

genes might be consistent with the increasing calcium content in root tissue of the two genotypes. However, the expression of CAX1 was observed to be low in root tissue as compared to those in other tissues as reported earlier (Carter et al., 2004; Conn and Gilliham, 2010; Cocozza et al., 2008). This suggests that although calcium uptake is taking place at high rate, it is not getting stored in the vacuole rather it is effluxed in the root apoplast and is trafficked to the cells by means of the water transpiration stream. The transporters TPC1 and ATPase might be expressed at the plasma membrane as suggested earlier (Hashimoto et al., 2005; Kurusu et al., 2005; Wang et al., 2005) and

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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3.2.5. In flag leaf The expression of all the transporter and regulatory genes was found to be downregulated in flag leaf (Fig. 2b), however, the calcium content, in flag leaf was highest (Fig. 2a) among all the tissues in both the genotypes. This indicates that the calcium accumulation in flag leaf is largely dependent on the transpirational pull and that isoforms, other than the ones identified in the present study are being expressed in flag leaf. Interestingly, the calcium content in flag leaves decreased at S3 (grain

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3.2.2. In stem tissue 359 High levels of expression and similar expression patterns were ob360 served for all the transporter genes and CaM protein kinases (Fig. 2d) 361 in the stem tissue corresponding with the high calcium content in finger 362 millet stem. Increased expression of TPC1 and CAX1 was observed in 363 both the genotypes with higher expression being recorded in GP-45. A 364 higher expression of TPC1 was however, observed at 90 DAS in GP-1 365 than in GP-45 which corresponds to higher calcium content in GP-1 366 stem at 90 DAS (Fig. 2c). Most interestingly, a thousand-fold increase 367 in expression was observed for ATPase gene from 30 DAS to 90 DAS in 368 Q15 both the genotypes with the highest expression at 90 DAS in GP-45. 369 This observation again indicates increased calcium supply at 90 DAS 370 for the developing reproductive tissue. The long distance transport of 371 calcium is proposed to be predominantly apoplastic and is dependent 372 upon the water flow generated due to transpiration. Metzner et al. 373 (2010) suggested the role of some active transport to pump the calcium 374 in the apoplast, to be taken up by phloem. The ATPase might be strongly 375 expressed in the xylem parenchyma cells and effluxing the symplastically 376 transported calcium (as suggested by high expression of calcium channel, 377 TPC1), up in the culm of the finger millet plant into the apoplast. 378 The expression levels and expression pattern of ATPase and CaMK1 379 were observed to be similar. Similarly, the expression pattern was alike 380 for TPC1 and CaMK2. This indicates the possible regulation of ATPase by 381 CaMK1 and that of TPC1 by CaMK2. Earlier inverse effect of intracellular 382 Ca2+ ion was reported on ACA2 gene via CaM and Ca2+-dependent pro383 tein kinase (CDPK) (Harmon et al., 2000; Harper et al., 1994; Hwang 384 et al., 2000). However, in finger millet the CaM dependent protein ki385 nases (CaMKs) suggest possible activation of the transporter. Expres386 sion of both CaM and 14-3-3 gene was again downregulated in stem 387 in both the genotypes.

3.2.4. In developing spikes Increasing calcium content is observed from the early S1 stage to the mature S4 stage (Fig. 2a). In the developing spike, all the transporters exhibited an increased expression from S1 to S4 stage with higher expression in GP-45 (Fig. 2b) and hence correspond to the higher calcium content observed. Higher expression of TPC1 and CAX1 genes in GP-45 indicates higher uptake and accumulation of calcium as compared to those in GP-1. In Arabidopsis, CAX1 expression peaks during the earlier stages of seed development whereas in later stages, CAX3 expression is higher (Punshon et al., 2012). However interestingly, in finger millet CAX1 expression was observed to be continuously increasing from the early S1 stage to late S4 stage, in both the genotypes. A high and increasing expression of Ca2+ ATPase was also observed in the two genotypes in contrast to the decreasing water flow to developing spike/seeds. In millets, xylem and phloem separate from each other in the pedicel of the spikelet (Zee and O'brien, 1971). Phloem consists of only few sieve elements associated with parenchyma cells and further removed from the ovule while xylem elements are reduced to one or two. The region of nutrient entry to the developing embryo and endosperm is restricted to a small gap in the seed coat lying at the base of the ovary enriched with cells of aleurone layer with wall in growths similar to transfer cells (Zee and O'brien, 1971). This explains the highest calcium accumulation in aleurone layer in finger millet seeds (Nath et al., 2013) and again suggesting that the Ca2+ ATPase might be expressed on the plasma membrane of xylem parenchyma cells, facilitating calcium unloading into the apoplast and TPC1 and other channels might facilitate calcium uptake by the aleurone cells and the developing embryo. A higher expression of Ca2+ ATPase gene was observed in GP-1 genotype than in GP-45. This indicates that a higher level is required to fulfill the demands of proper growth in the low calcium accumulating genotype GP-1. All the regulatory proteins also exhibited expression patterns similar to the transporter genes with generally a higher expression in GP-45 genotype (Fig. 2b). CaM exhibited a similar expression pattern as Ca2+ ATPase gene indicating that this CaM isoform might be specific to developing spike and activating the Ca2+ ATPase through binding to the CBD domain (Amtmann and Blatt, 2009). CaMK1 also exhibited an expression pattern similar to Ca2+ ATPase however, the expression was low in developing spike. The expression pattern of CaMK2 was again observed to be similar to TPC1. 14-3-3 gene exhibited a similar expression pattern to CAX1 raising the possibility of CAX1 regulation through 14-33 binding.

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vacuoles, except for signaling, calcium is not mobilized to apoplast or to other tissues. No significant difference in expression was observed for ATPase (except at 90 DAS in GP-1) and calcium channel TPC1. These observations indicate that other isoforms of these transporters might be expressed in leaf tissue and supports the fact that different isoforms of transporter genes and regulatory proteins are present in plants with different spatial and temporal expression. Intriguingly, the expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf tissue (Fig. 2d). 14-3-3 is a large gene family of regulatory proteins found in all eukaryotes and is involved in many cellular processes (Aitken, 1996) with targets as diverse as metabolic enzymes (Comparot et al., 2003; Huber et al., 2002), transcription factors (Schultz et al., 1998), protein kinases and ion transporters (de Boer, 2002). This raise a possibility that 14-3-3 might interact with the CAX1 through the regulatory R-domain.

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facilitating respectively the uptake of calcium into the cytoplasm and its delivery to the apoplast as hypothesized by White and Broadley (2003). CaM stimulates the autoinhibited ATPase (Medvedev, 2005). CaM and Ca2+-dependent protein kinases (CDPK) exert regulatory effect on Ca2+ type IIB pumps. At low calcium concentrations in the cytosol, CPK1 inhibits the activity of Ca2+-ATPase (Hwang et al., 2000). As calcium concentration increases, CaM is activated and binds to the autoinhibitory domain of the Ca2+ pump, thereby releasing the inhibition caused by CDPK. Here, CaM expression was observed to be downregulated indicating that the isolated isoform might not be functional in root tissue. Both the CaM dependent protein kinases, CaMK1 and CaMK2 were expressed in the root tissue with maximum expression at 30 DAS and CaMK1 being highly up-regulated in GP-45 genotype. However, the expression patterns of CaMK1 and CaMK2 don't correspond with the expression of any of the transporter genes in root tissue. The expression of 14-3-3 gene was also downregulated in root tissue for both the genotypes.

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3.2.3. In leaf tissue The calcium delivery and distribution in leaves are mostly dependent 390 on the pathway of water flow to and through the leaves (Gilliham et al., 391 2011). The calcium content in finger millet leaves was higher than those 392 in the root and stem tissue (Fig. 2c) with higher content in GP-45 leaves 393 Q16 as compared to GP-1. The leaf area was observed to be higher for GP-45 394 genotype. This indicates that higher transpiration might attribute to 395 higher calcium accumulation in GP-45 genotype. ATPase and CAX pro396 teins have been implicated with a role in calcium accumulation within 397 the leaf and localization of the protein in mesophyll tonoplast prepara398 tions and might contribute to the higher accumulation of Ca2+ ions in 399 Q17 the mesophyll (Geisler et al., 2000; Lee et al., 2007; Yang et al., 2008). 400 Over-expression of an N-terminally truncated form of AtCAX1 (called 401 sCAX1) was associated with increased leaf calcium (Park et al., 2005). 402 An increased expression of CAX1 gene was observed at 60 DAS with 403 higher expression in GP-45 genotype. Even though the expression of 404 CAX1 decreases, calcium content increased since, once stored in the

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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However, over-expression of sCAX1 leads to calcium-deficiency symptoms (Park et al., 2005). The finger millet CAX1 can thus be a potential candidate gene for increasing the calcium content in developing seeds and other edible tissues. The CaM isoform from finger millet was specifically found to have strong expression in developing spike particularly in high calcium genotype. Therefore, this CaM isoform can be utilized to stimulate the type IIB Ca2 + ATPase specifically in the developing seeds. A CAX–CaM dual gene construct may be designed for targeted co-expression under grain endosperm cell-specific promoters to fortify cereals with bioavailable calcium. The strong expression of CaM stimulated type IIB Ca2+ ATPase gene in the root and developing spike and staggeringly high expression in the stem signify its role in calcium translocation from source to sink. Comparison of the calcium ATPase sequence with that from finger millet might provide some genomic elements to be introduced or modified accordingly to increase its activity. A single gene might do the trick of enhancing the source to sink relationship. Studies on finger millet CAX1 and ATPase might offer further insights in understanding their autoinhibition and strategic regulation. The finger millet plant might as well befall as a model system for better understanding of the underlying genetic control and molecular physiological mechanisms contributing to high grain calcium.

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filling) stage in both the genotypes. The senescing flag leaf re-allocates various minerals and proteins from the source to the sink during grain filling (Sperotto et al., 2009; Verma et al., 2004). The results suggest the presence of some transporters facilitating the mobilization of calcium. The relatively lower concentration of calcium in flag leaf of GP-45 compared to that of GP-1 indicates the faster mobilization of calcium in high grain calcium accumulating genotype from source to sink than low grain calcium genotype probably due to activation/up-regulation of some transporter genes. In brief, all the genes express differentially (Fig. 3) leading to differential spatial and temporal calcium accumulation in the two genotypes. The genotype GP-45 accumulates more calcium in seeds while GP-1 accumulates more in flag leaf. The possibility of remobilization of calcium from flag leaf as indicated in the present study remains to be seen. It is generally believed that due to low transpiration and low density of stomata, the non-exposed tissues including developing seeds are low in calcium. However in the present study, we have found that calcium content and CAX1 expression increased continuously from the early (S1 stage) to late (S4 stage) seed development stage with higher expression in the high calcium genotype GP-45. CAX proteins have earlier been assigned a role in calcium accumulation within the leaf mesophyll (Cheng et al., 2005; Hirschi, 1999; Hirschi et al., 2000).

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Fig. 3. Depiction of the role of the potential transporters and their regulatory genes during translocation of calcium from the rhizosphere by the (1) root and root hair and translocation along with the xylem stream through (2) stem, distribution to the (3) leaves and phloem loading for movement into the (4) flag leaf and (5) developing spike (6). The redistribution of Ca from flag leaf like other minerals is possible or not remains to be seen. The up (↑) and down (↓) regulation of these genes are given in the sketch diagram.

Please cite this article as: Mirza, N., et al., Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.005

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The authors wish to acknowledge the Department of Biotechnology, Govt. of India for providing financial support in the form of Programme Support for research and development in Agricultural Biotechnology at G.B. Pant University of Agriculture and Technology, Pantnagar (Grant No. BT/PR7849/AGR/02/2006).

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.005.

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